Compound, phosphorylation inhibitor, insulin resistance improving agent, preventive or therapeutic agent for diabetes, and screening method

ABSTRACT

A new compound inhibiting phosphorylation of Ser727 of STAT3, a phosphorylation inhibitor containing the new compound, an insulin resistance improving agent and a preventive or therapeutic agent for diabetes; and a screening method for at least one of the insulin resistance improving agent and the preventive or therapeutic agent for diabetes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.13/214,712, filed on Aug. 22, 2011, which in turn is a continuationapplication of PCT/JP2010/052765, filed on Feb. 23, 2010, and whichclaims priority from Japanese patent application No. 2009-039997, filedFeb. 23, 2009, the entire contents of which are incorporated herein byreference.

FIELD

The embodiments discussed herein relate to a new compound having aninhibitory activity against phosphorylation of Ser727 of STAT3, aphosphorylation inhibitor containing the new compound, an insulinresistance improving agent, a preventive or therapeutic agent fordiabetes, and an efficient screening method for at least one of theinsulin resistance improving agent and the preventive or therapeuticagent for diabetes.

BACKGROUND

The blood glucose level is constantly controlled so as to fall within acertain range at a normal state. A peptide hormone called insulin isimportant for controlling the blood glucose level. This peptide hormoneis excreted from 13 cells of islets of Langerhans present in thepancreas. When the insulin binds to an insulin receptor present on thecell membrane, various signal transductions occur to suppressgluconeogenesis, leading to a drop in the blood glucose level.

The reason why the blood glucose level is controlled is that glucoseserves as a harmful substance causing glycosylation stress in tissuealthough it is a main energy source for organs including the brain.

When the control ability of the blood glucose level (glucose tolerance)is decreased, one is in a state where the blood glucose level haspathologically increased or a state where the blood glucose levelpotentially increases. Such a state that the glucose metabolism has beenabnormal is diabetes.

The diabetes is classified into type 1 diabetes (insulin-dependentdiabetes) and type 2 diabetes (insulin-independent diabetes). The type 2diabetes, most of the diabetes belong to, is further classified intodiabetes caused due to a decrease in the level of excreted insulin anddiabetes in which although insulin is excreted at a sufficiently highlevel, the blood glucose level does not decrease due to reduction ininsulin susceptibility of glucose in the target cells. The latter caseis called insulin resistance.

The current diabetes therapy is conducted using various drugs selecteddepending on the pathological conditions. The types of the drugs areclassified roughly into insulin, an insulin-excretion promoter, aglucose absorption inhibitor and an insulin resistance improving agent.

For patients exhibiting the above-described insulin resistance, aninsulin resistance improving agent such as metformin hydrochloride,buformine hydrochloride or pioglitazone hydrochloride is used as thefirst-line drug. However, such an insulin resistance improving agentinvolves gastrointestinal disorder as side effects. In addition, itcannot be applied to those having a history of heart failure. Also,pioglitazone hydrochloride involves increase in body weight as sideeffects, which imposes problematic burden on patients who are receivingdietary therapy.

Meanwhile, besides insulin, signal transducer and activator oftranscription 3 (STAT3) has recently been reported as a factor thatdecreases the blood glucose level (see Inoue H et al., Nat Med, 10(2),2004, p. 168-74).

The STAT3 is a protein that works for both of signal transduction andtranscription activation, and controls processes such as cell growth,differentiation and survival. The STAT3 exists in the cytoplasm in thenon-phosphorylated form. When the STAT3 is activated by Janus kinase(JAK) so that its Tyr705 is phosphorylated, a homodimer of the STAT3 isformed and transferred into the nucleus, where it serves as atranscriptional factor to activate the target gene.

The homodimer of the STAT3 serves in the nucleus as an antagonistagainst a transcriptional factor responsible for gluconeogenesis.Increase in the expression level thereof suppresses gluconeogenesis toreduce the blood glucose level.

Also, as has been known, when Ser727 of the STAT3 is phosphorylated byextracellular signal-regulated kinase 2 (ERK2), formation of thehomodimer of the STAT3 is inhibited (see Chung J et al., Mol Cell Biol,17(11), 1997, p. 6508-16; Jain N et al., Oncogene, 17(24), 1998, p.3157-67; and Liu S et al., Proc Natl Acad Sci USA, 103(14), 2006, p.5326-31).

However, diabetes therapeutic drugs relating to the STAT3 have not beenknown.

Thus, in the prevention or therapy for diabetes, there have not yet beenprovided an effective, safe drug having a target molecule and mechanismwhich are different from those of these existing drugs, and an efficientscreening method for a candidate substance of the above drug. Atpresent, keen demand has arisen for the provision of them.

SUMMARY

According to an aspect of an embodiment, a compound inhibitsphosphorylation of Ser727 of STAT3.

According to another aspect of an embodiment, a phosphorylationinhibitor contains the above compound.

According to still another aspect of an embodiment, an insulinresistance improving agent contains the above phosphorylation inhibitor.

According to yet another aspect of an embodiment, a preventive ortherapeutic agent for diabetes contains the above insulin resistanceimproving agent.

According to even another aspect of an embodiment, a screening methodfor at least one of the insulin resistance improving agent and thepreventive or therapeutic agent for diabetes includes determining thepresence or absence of at least interaction between STAT3 and ERK2 toselect a compound having a phosphorylation inhibitory activity andevaluating the compound for whether X>Y where X denotes a HOMA-IR valueof a diabetes model animal to which the compound has not beenadministered and Y denotes a HOMA-IR value of a diabetes model animal towhich the compound has been administered.

The object and advantages of the invention will be realized and attainedby means of the elements and combinations particularly pointed out inthe claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and arenot restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 indicates the results of a phosphorylation inhibitory activitytest performed by western blotting.

FIG. 2 is an enlarged view of FIG. 1.

FIG. 3 is a graph of the fasting blood glucose levels measured in anefficacy test on Day 0, Day 14 and Day 28 after administration, whereinthe five bars corresponding to each day indicate, from the left to theright, a bar for a solvent control group, a bar for a 150 mg/kgmetformin administration group, a bar for a 4.00 mg/kg peptideadministration group, a bar for a 1.33 mg/kg peptide administrationgroup and a bar for a 0.44 mg/kg peptide administration group.

FIG. 4 is a graph of the postprandial blood glucose levels measured inan efficacy test on Day 1, Day 13 and Day 27 after administration,wherein the five bars corresponding to each day indicate, from the leftto the right, a bar for a solvent control group, a bar for a 150 mg/kgmetformin administration group, a bar for a 4.00 mg/kg peptideadministration group, a bar for a 1.33 mg/kg peptide administrationgroup and a bar for a 0.44 mg/kg peptide administration group.

FIG. 5 is a graph of the fasting blood insulin levels measured in anefficacy test on Day 0 and Day 28 after administration, wherein the fivebars corresponding to each day indicate, from the left to the right, abar for a solvent control group, a bar for a 150 mg/kg metforminadministration group, a bar for a 4.00 mg/kg peptide administrationgroup, a bar for a 1.33 mg/kg peptide administration group and a bar fora 0.44 mg/kg peptide administration group.

FIG. 6 is a graph of the HOMA-IR values measured in an efficacy test onDay 0 and Day 28 after administration, wherein the five barscorresponding to each day indicate, from the left to the right, a barfor a solvent control group, a bar for a 150 mg/kg metforminadministration group, a bar for a 4.00 mg/kg peptide administrationgroup, a bar for a 1.33 mg/kg peptide administration group and a bar fora 0.44 mg/kg peptide administration group.

FIG. 7 is a graph of increments of the blood glucose levels measured inan efficacy test, wherein the five bars corresponding to each day afteradministration indicate, from the left to the right, a bar for a solventcontrol group, a bar for a 150 mg/kg metformin administration group, abar for a 4.00 mg/kg peptide administration group, a bar for a 1.33mg/kg peptide administration group and a bar for a 0.44 mg/kg peptideadministration group.

FIG. 8 is a graph of increments of the fasting blood insulin levelsmeasured in an efficacy test, wherein the five bars corresponding toeach day after administration indicate, from the left to the right, abar for a solvent control group, a bar for a 150 mg/kg metforminadministration group, a bar for a 4.00 mg/kg peptide administrationgroup, a bar for a 1.33 mg/kg peptide administration group and a bar fora 0.44 mg/kg peptide administration group.

DESCRIPTION OF EMBODIMENTS (New Compound)

The disclosed new compound inhibits phosphorylation of Ser727 of theSTAT3.

The STAT3 has several phosphorylation sites. For example, when itsSer727 is phosphorylated, the STAT3 cannot form its homodimer to betransferred into the nucleus. Thus, it cannot serve as a transcriptionalfactor.

The Ser727 of the STAT3 is phosphorylated by ERK2, for example.Specifically, the ERK2 is activated through phosphorylation of itsTyr185 by MAPK/ERK kinase 1 (MEK1) and then binds to the STAT3 to form aheterodimer, where the Ser727 of the STAT3 is phosphorylated.

Thus, when the phosphorylation of the Ser727 is inhibited by the abovephosphorylation inhibitor, the STAT3 is not prevented from forming itshomodimer and the formed homodimer of the STAT3 can serve as atranscriptional factor.

<New Compound Containing Peptide Represented by Amino Acid SequenceStructure (I)>

The above new compound contains at least a peptide represented by thefollowing amino acid sequence structure (I), for example.

—Peptide Represented by Amino Acid Sequence Structure (I)—

The peptide represented by the amino acid sequence structure (I) is asfollows.

α-α′-β-γ-δ′-σ-δ . . . Amino acid sequence structure (I)

The peptide represented by the amino acid sequence structure (I) ispreferably at least one of peptides represented by the following aminoacid sequence structures (II) and (III).

Lys-Lys-β-γ-δ′-σ-δ . . . Amino acid sequence structure (II)

α-α′-β-γ-Leu-σ-Leu . . . Amino acid sequence structure (III)

In the amino acid sequence structures (I), (II) and (III), a denotes anN terminus, δ denotes a C terminus, α and α′ each denote lysine orarginine, δ and δ′ each denote leucine or isoleucine, and β, γ and σeach denote any amino acid.

The amino acid denoted by β is not particularly limited and may beappropriately selected depending on the intended purpose. It ispreferably tyrosine.

The amino acid denoted by γ is not particularly limited and may beappropriately selected depending on the intended purpose. It ispreferably isoleucine.

The amino acid denoted by σ is not particularly limited and may beappropriately selected depending on the intended purpose. It ispreferably alanine.

Among the peptides represented by the amino acid sequence structures(I), (II) and (III), more preferred is a peptide containing an aminoacid sequence expressed by the following SEQ ID No. 1. Particularlypreferably is a peptide itself expressed by the following SEQ ID No. 1.

(SEQ ID No. 1) Lys-Lys-Tyr-Ile-Leu-Ala-Leu

So long as the above new compound has an inhibitory activity againstphosphorylation of the Ser727 of the STAT3, the above peptide may havean amino acid sequence expressed by the above SEQ ID No. 1 in all orpart of which one to several amino acids have been substituted or added.Also, the peptide may be chemically modified at its N or C terminus.This chemical modification is not particularly limited and may beappropriately selected depending on the intended purpose. Examplesthereof include acetylation, myristoylation and amidation.

The amount of the peptide represented by the above amino acid sequencestructure (I) contained in the new compound is not particularly limitedand may be appropriately selected depending on the intended purpose.Also, the new compound may be the peptide itself represented by theabove amino acid sequence structure (I).

The method for obtaining the peptide is not particularly limited and maybe appropriately selected depending on the intended purpose. Examplesthereof include a method in which the peptide is obtained as a syntheticpeptide produced through chemical synthesis.

The method for chemically synthesizing the peptide is not particularlylimited and may be appropriately selected depending on the intendedpurpose. Examples thereof include a method in which the peptide ischemically synthesized using a peptide synthesizer (product of ShimadzuCorporation).

The above new compound may be in the form of salt. The salt is notparticularly limited and may be appropriately selected depending on theintended purpose. Examples thereof include carboxylates, inorganic acidsalts, amino acid salts and sulfonates.

Examples of the carboxylates include trifluoroacetate, acetate,trichloroacetate, hydroxyacetate, lactate, citrate, tartarate, oxalate,benzoate, butyrate, maleate, propionate, formate and malate.

Examples of the inorganic acid salts include hydrohalic acid salts,sulfate, nitrate, phosphate and carbonate.

Examples of the amino acid salts include alginate, aspartate andglutamate.

Examples of the sulfonates include methanesulfonate andp-toluenesulfonate.

(Phosphorylation Inhibitor)

The disclosed phosphorylation inhibitor contains at least theabove-described new compound or a salt thereof; and, if necessary,further contains other ingredients.

The amount of the new compound or a salt thereof contained the abovephosphorylation inhibitor is not particularly limited and may beappropriately selected depending on the intended purpose. Also, thephosphorylation inhibitor is the new compound or a salt thereof itself.

<Other Ingredients>

The other ingredients contained in the phosphorylation inhibitor are notparticularly limited and may be appropriately selected depending on theintended purpose. Examples thereof include pharmacologically acceptablecarriers.

The above carriers are not particularly limited and may be appropriatelyselected depending on, for example, the dosage form of thephosphorylation inhibitor.

The amount of the other ingredients contained in the phosphorylationinhibitor is not particularly limited and may be appropriately selecteddepending on the intended purpose.

<Measurement Method for Phosphorylation Inhibitory Activity>

The measurement method for the phosphorylation inhibitory activity isnot particularly limited and may be appropriately selected depending onthe intended purpose. Examples thereof include immunoassay.

The immunoassay is not particularly limited and may be appropriatelyselected depending on the intended purpose. Examples thereof includeimmunostaining, immunoprecipitation, western blotting and ELISA, withwestern blotting being preferred.

<Application>

The application of the phosphorylation inhibitor is not particularlylimited and can suitably used as a drug such as the below-describedinsulin resistance improving agent or preventive or therapeutic agentfor diabetes. Also, the phosphorylation inhibitor can be used as, forexample, a reagent for assay using phosphorylation as an index.

When the phosphorylation inhibitor is used as a reagent, the method forusing it is not particularly limited and may be appropriately selecteddepending on the intended purpose. For example, the phosphorylationinhibitor may be added to a culture liquid of culture cells.

(Insulin Resistance Improving Agent and Preventive or Therapeutic Agentfor Diabetes) <Insulin Resistance Improving Agent>

The disclosed insulin resistance improving agent contains at least theabove-described phosphorylation inhibitor; and, if necessary, furthercontains other ingredients.

The amount of the phosphorylation inhibitor contained in the insulinresistance improving agent is not particularly limited and may beappropriately selected depending on the intended purpose. Also, theinsulin resistance improving agent may be the phosphorylation inhibitoritself.

—Evaluation of Insulin Resistance—

The method for evaluating whether the insulin resistance improving agentimproves insulin resistance is not particularly limited and may beappropriately selected depending on the intended purpose. Preferred is amethod of evaluating it using the HOMA-IR (homeostasis model assessment)method. The HOMA-IR method is a method of calculating a HOMA-IR valueserving as an index of insulin resistance using the followingcalculation formula.

HOMA-IR (index of insulin resistance)=fasting insulin level(μU/mL)×fasting blood glucose level (mmol/L)/22.5

This evaluation is based on that the lower the HOMA-IR value is, themore improved the insulin resistance is (see Matthews D R et al.,Diabetologia 1985 July 28(7), 412-9, Kanauchi M et al., Diabetes Care,October 25(10), 2002, p. 1891-2).

The fasting is not particularly limited and may be appropriatelyselected depending on the intended purpose. It is preferably 12 hours to14 hours after eating.

The method for obtaining a blood sample used for measuring the fastinginsulin level and the fasting blood glucose level is not particularlylimited. The blood sample can be obtained by routinely-used bloodsampling. The blood sample is not particularly limited and may beappropriately selected depending on the intended purpose. It ispreferably a serum. The method for preparing the serum is notparticularly limited and may be appropriately selected depending on theintended purpose. Examples thereof include a method in which the sampledblood is centrifuged.

The method for determining the fasting insulin level is not particularlylimited and may be appropriately selected depending on the intendedpurpose. Examples thereof include a method in which the fasting insulinlevel is determined using a mouse insulin ELISA kit (Cat. #EZRMI-13K,product of Linco Research Inc.).

The method for determining the fasting blood glucose level is notparticularly limited and may be appropriately selected depending on theintended purpose. Examples thereof include a method in which the fastingblood glucose level is determined using Johnson one-touch Ultra GlucoseMonitoring System (product of Johnson & Johnson K.K.).

—Application—

As to the application of the insulin resistance improving agent, theinsulin resistance improving agent can suitably used as a drug such asthe below-described preventive or therapeutic agent for diabetes.

<Preventive or Therapeutic Agent for Diabetes>

The disclosed preventive or therapeutic agent for diabetes contains atleast the insulin resistance improving agent; and, if necessary, furthercontains other ingredients.

The amount of the insulin resistance improving agent contained in thepreventive or therapeutic agent for diabetes is not particularly limitedand may be appropriately selected depending on the intended purpose.Also, the preventive or therapeutic agent for diabetes may be theinsulin resistance improving agent itself.

<Other Ingredients>

The other ingredients contained in the insulin resistance improvingagent or the preventive or therapeutic agent for diabetes are notparticularly limited and may be appropriately selected depending on theintended purpose. Examples thereof include pharmacologically acceptablecarriers.

The above carriers are not particularly limited and may be appropriatelyselected depending on, for example, the dosage form of thephosphorylation inhibitor, the insulin resistance improving agent or thepreventive or therapeutic agent for diabetes.

The amount of the other ingredients contained in the insulin resistanceimproving agent or the preventive or therapeutic agent for diabetes isnot particularly limited and may be appropriately selected depending onthe intended purpose.

(Use)

The phosphorylation inhibitor, the insulin resistance improving agent,and the preventive or therapeutic agent for diabetes may be used aloneor in combination. Also, they are used in combination with a drugcontaining other active ingredients. Furthermore, the phosphorylationinhibitor, the insulin resistance improving agent, and the preventive ortherapeutic agent for diabetes may be formulated into a drug containingother active ingredients before use.

(Dosage Form)

The dosage form of the phosphorylation inhibitor, the insulin resistanceimproving agent, or the preventive or therapeutic agent for diabetes isnot particularly limited and may be appropriately selected depending ona desired administration method. Examples thereof include an oral solidpreparation, an oral liquid preparation, an inhalation powder and aninjection. Among them, an injection is preferred since it is notdigested more easily than an oral solid preparation, an oral liquidpreparation or an inhalation powder is.

<Injection>

The injection is not particularly limited and may be appropriatelyselected depending on the intended purpose. Examples of the injectioninclude a solution, a suspension and a solid preparation reconstitutedupon use.

The method for producing the injection is not particularly limited andmay be a routine method. For example, the injection can be produced byadding a pH adjuster, a buffer, a stabilizing agent, a tonicity agent, alocal anesthetic, etc. to the phosphorylation inhibitor, the insulinresistance improving agent, or the preventive or therapeutic agent fordiabetes. Here, the pH adjuster or buffer is not particularly limitedand may be appropriately selected depending on the intended purpose.Examples of the pH adjuster or buffer include sodium citrate, sodiumacetate and sodium phosphate. The stabilizing agent is not particularlylimited and may be appropriately selected depending on the intendedpurpose. Examples of the stabilizing agent include sodium pyrosulfite,EDTA, thioglycolic acid and thiolactic acid. The tonicity agent is notparticularly limited and may be appropriately selected depending on theintended purpose. Examples of the tonicity agent include sodium chlorideand glucose. The local anesthetic is not particularly limited and may beappropriately selected depending on the intended purpose. Examples ofthe local anesthetic include procaine hydrochloride and lidocainehydrochloride.

<Administration>

In the phosphorylation inhibitor, the insulin resistance improvingagent, or the preventive or therapeutic agent for diabetes, theadministration method, the administration dose, the time ofadministration and the subject to be administered are not particularlylimited and may be appropriately selected depending on the intendedpurpose.

The administration method is not particularly limited and may beappropriately selected depending on the intended purpose. Examples ofthe administration method include oral administration, inhalation andinjection, with injection being preferred. The administration dose isnot particularly limited and may be appropriately selected consideringvarious factors of a subject to be administered, such as the age, bodyweight, constitution, symptom and the presence or absence ofadministration of a drug containing other active ingredients.

The animal species serving as the subject to be administered is notparticularly limited and may be appropriately selected depending on theintended purpose. Examples of the animal species include human, monkey,pig, bovine, sheep, goat, dog, cat, mouse, rat and bird. Among them,they are suitably administered to human.

(Production Method)

The production method for the phosphorylation inhibitor, the insulinresistance improving agent, or the preventive or therapeutic agent fordiabetes is not particularly limited and may be appropriately selecteddepending on, for example, an intended dosage form described above.

(Screening Method)

The disclosed screening method for at least one of the insulinresistance improving agent and the preventive or therapeutic agent fordiabetes includes selecting a compound having at least a phosphorylationinhibitory activity and evaluating the compound based on a HOMA-IR valueof a diabetes model animal to which the compound has been administered;and, if necessary, further include other steps.

<Selection>

The selection is performed by measuring at least the presence or absenceof interaction between STAT3 and ERK2 to select a compound having aphosphorylation inhibitory activity, and includes a mixture preparationstep, an antibody reaction step and a screening step.

—Mixture Preparation Step—

The mixture preparation step is a step of preparing a mixture by, forexample, mixing together at least a compound, STAT3 whose Ser727 is notphosphorylated, and an enzyme that phosphorylates the STAT3. This stepfurther includes a treatment by which the STAT3 phosphorylation enzymecontained in the mixture is allowed to phosphorylate the Ser727 of theSTAT3.

—Mixture—

The mixture contains at least the above compound, the STAT3 whose Ser727is not phosphorylated, and the enzyme that phosphorylates the STAT3;and, if necessary, further contains other ingredients.

—Compound—

The compound contained in the mixture is not particularly limited andmay be appropriately selected depending on the intended purpose.Examples thereof include peptides.

The method for obtaining the compound is not particularly limited andmay be appropriately selected depending on the intended purpose. Forexample, it may be obtained by chemical synthesis or gene recombinanttechniques.

The chemical synthesis or gene recombinant technique used for obtainingthe compound is not particularly limited and may be appropriatelyselected depending on the intended purpose from, for example, knownmethods in the art.

The amount of the compound contained in the mixture is not particularlylimited and may be appropriately selected depending on the intendedpurpose. For example, it is preferably 0.1 mM to 50 mM as a finalconcentration, more preferably 0.5 mM to 5 mM.

The method for preparing the compound is not particularly limited andmay be appropriately selected depending on the intended purpose.Examples thereof include a method in which the compound is preparedusing a solution containing dimethylsulfoxide (DMSO).

The final concentration of the DMSO is not particularly limited and maybe appropriately selected depending on the intended purpose. It ispreferably 1 mM to 100 mM, more preferably 5 mM to 15 mM.

—STAT3—

The method for obtaining the STAT3 whose Ser727 is not phosphorylated(hereinafter may be referred to as “non-phosphorylated STAT3”) is notparticularly limited and may be appropriately selected depending on theintended purpose. For example, the non-phosphorylated STAT3 may be acommercially available product.

The amount of the non-phosphorylated STAT3 contained in the mixture isnot particularly limited and may be appropriately selected depending onthe intended purpose. For example, it is preferably 0.01 μg/mL to 0.5μg/mL as a final concentration, more preferably 0.025 μg/mL to 0.075μg/mL.

—Enzyme that Phosphorylates STAT3—

The enzyme that phosphorylates the STAT3 refers to an enzyme thatphosphorylates the Ser727 of the non-phosphorylated STAT3.

The enzyme that phosphorylates the STAT3 is not particularly limited andmay be appropriately selected depending on the intended purpose. Forexample, it is preferably ERK2 that has been activated throughphosphorylation of its Tyr185 (hereinafter may be referred to as“activated ERK2”).

The method for obtaining the activated ERK2 is not particularly limitedand may be appropriately selected depending on the intended purpose. Forexample, it may be a commercially available product.

The amount of the activated ERK2 contained in the mixture is notparticularly limited and may be appropriately selected depending on theintended purpose. For example, it is preferably 0.0001 μg/mL to 0.015μg/mL as a final concentration, more preferably 0.0005 μg/mL to 0.005μg/mL.

—Other Ingredients—

The other ingredients are not particularly limited and may beappropriately selected depending on the intended purpose. Examplesthereof include a reaction buffer in which the activated ERK2 canexhibit its enzymatic activity, ATP, magnesium chloride and DMSO.

The composition of the reaction buffer is not particularly limited andmay be appropriately selected depending on the intended purpose. Thereaction buffer preferably has the following composition: 50 mM tris-HCl(pH 7.5), 10 mM magnesium chloride, 0.02% by mass bovine serum albumin(BSA) and 1 mM dithiothreitol (DTT) (these concentrations are all finalconcentrations).

The concentration of the ATP is not particularly limited and may beappropriately selected depending on the intended purpose. It ispreferably 0.1 mM to 5 mM as a final concentration, more preferably 0.5mM to 2 mM.

The concentration of the magnesium chloride is not particularly limitedand may be appropriately selected depending on the intended purpose. Itis preferably 1 mM to 100 mM as a final concentration, more preferably 5mM to 20 mM.

The concentration of the DMSO is not particularly limited and may beappropriately selected depending on the intended purpose. It ispreferably 1% by mass to 25% by mass as a final concentration, morepreferably 5% by mass to 22% by mass.

—Preparation of Mixture—

The preparation method for obtaining the mixture is not particularlylimited and may be appropriately selected depending on the intendedpurpose. Examples thereof include a method in which the mixture issuspended with a pipette.

The timing when the compound, the non-phosphorylated STAT3, the enzymethat phosphorylates the STAT3, or the other ingredients are added is notparticularly limited. They may be added to the mixture at an appropriatetiming during the preparation of the mixture.

—Treatment for Phosphorylation—

The treatment by which the enzyme that phosphorylates the STAT3 isallowed to phosphorylate the Ser727 of the non-phosphorylated STAT3(hereinafter may be referred to as “phosphorylation treatment”) is notparticularly limited and may be appropriately selected depending on theintended purpose.

The temperature at the phosphorylation treatment is not particularlylimited and may be appropriately selected depending on the intendedpurpose. It is preferably 20° C. to 30° C.

The period of the phosphorylation treatment is not particularly limitedand may be appropriately selected depending on the intended purpose. Itis preferably 30 min to 150 min.

The method for terminating the phosphorylation is not particularlylimited and may be appropriately selected depending on the intendedpurpose.

Examples thereof include a method in which ethylenediamine tetraacetateis added to the mixture.

—Antibody Reaction Step—

The antibody reaction step is, for example, a step of reacting the STAT3whose Ser727 has been phosphorylated in the mixture with an antibodythat binds to a phosphorylated site of the STAT3 whose Ser727 has beenphosphorylated. This step includes a detection treatment for the boundantibody.

—Antibody that Binds to Phosphorylated Site of Phosphorylated STAT3—

The antibody that binds to the phosphorylated site of the Ser727 of theSTAT3 (hereinafter may be referred to as “primary antibody”) is notparticularly limited, so long as it can recognize the phosphorylatedsite of the Ser727 of the STAT3, and may be appropriately selecteddepending on the intended purpose.

The primary antibody is not particularly limited and may beappropriately selected depending on the intended purpose. It may be, forexample, anti-pS727 STAT (product of SANTA CRUZ Inc., Cat. No.:sc-21876).

—Reaction—

The reaction is, for example, a reaction for allowing the primaryantibody (i.e., the antibody that binds to the phosphorylated site ofthe Ser727 of the STAT3) to bind to the STAT3 whose Ser727 has beenphosphorylated.

The temperature for the reaction between the STAT3 and the primaryantibody is not particularly limited and may be appropriately selecteddepending on the intended purpose. It is preferably 20° C. to 30° C.

The time of the reaction between the STAT3 and the primary antibody isnot particularly limited and may be appropriately selected depending onthe intended purpose. It is preferably 5 min to 15 min.

Here, the STAT3 with which the primary antibody reacts is the STAT3 inwhich the phosphorylation site of the Ser727 has been phosphorylated.

—Detection Treatment—

The detection treatment is, for example, a treatment for detecting thereacted STAT3.

The method for detecting the reacted STAT3 is not particularly limitedand may be appropriately selected depending on the intended purpose.Examples thereof include a detection method using an antibody that bindsspecifically to the primary antibody (hereinafter may be referred to as“secondary antibody).

The secondary antibody is not particularly limited and may beappropriately selected depending on the intended purpose. It may be, forexample, IgG of a host with which the primary antibody is produced.

Also, the secondary antibody is preferably labeled. The label is notparticularly limited and may be appropriately selected depending on theintended purpose. Examples thereof include horseradish peroxidase (HRP).

The method for detecting the HRP is not particularly limited and may beappropriately selected depending on the intended purpose. Examplesthereof include a method using a substrate of the HRP.

The substrate of the HRP is not particularly limited and may beappropriately selected depending on the intended purpose. Examplesthereof include dimethylformamide (DMF).

—Screening Step—

The screening step is, for example, a step of screening for the compoundpresent in the mixture containing the STAT3 to which the antibody hasnot bound, as a compound having a phosphorylation inhibitory activity.Specifically, in this step, the presence or absence of thephosphorylation inhibitory activity of the compound is determined and,when the compound has the phosphorylation inhibitory activity, thedegree of the phosphorylation inhibitory activity is determined.

The method for the screening is not particularly limited and may beappropriately selected depending on the intended purpose. Examplesthereof include a method in which the presence or absence of thephosphorylation inhibitory activity is determined based on the intensityof a band detected through western blotting (i.e., a band of the STAT3whose Ser727 has been phosphorylated).

The method for determining the presence or absence of thephosphorylation inhibitory activity based on the intensity of the bandis not particularly limited and may be appropriately selected dependingon the intended purpose. For example, the following method using as acontrol a mixture that does not contain the compound can be employed.Specifically, by comparing the mixture containing the compound with thecontrol in terms of the band intensity, the compound contained in themixture whose band is lighter than that of the control is determined asthe compound having the phosphorylation inhibitory activity.Alternatively, the following method using no control can be employed.Specifically, by comparing the mixtures containing the compounds withone another in terms of the band intensity, the compound contained inthe mixture whose band is lighter than those of the other mixtures isdetermined as having the phosphorylation inhibitory activity, and thiscompound is determined as a compound having a higher phosphorylationinhibitory activity.

The method for comparing the band intensities is not particularlylimited and may be appropriately selected depending on the intendedpurpose. Examples thereof include a method in which the bands arevisually compared with one another and a method in which the bands arecompared with one another using a device such as a densitometer.

<Evaluation>

The evaluation is performed by evaluating whether X>Y where X denotes aHOMA-IR of a diabetes model animal to which the compound has not beenadministered and Y denotes a HOMA-IR of a diabetes model animal to whichthe compound has been administered.

The animal species of the diabetes model animal is not particularlylimited and may be appropriately selected depending on the intendedpurpose. Examples thereof include monkey, pig, bovine, sheep, goat, dog,cat, mouse, rat and bird. Among them, mouse is preferred since screeningfor at least one of the insulin resistance improving agent and thepreventive or therapeutic agent for diabetes can easily be performed.The diabetes model mouse is not particularly limited and may beappropriately selected depending on the intended purpose. Examplesthereof include db/db mouse (genetically obese mice) (product ofShanghai SLAC Laboratory Animal).

As described above, it can be recognized that the lower the HOMA-IRvalue is, the more improved the insulin resistance is. Thus, when X>Y asa result of comparison between the HOMA-IR value (X) of the diabetesmodel mouse to which the compound has not been administered (serving asa control) and the HOMA-IR value (Y) of the diabetes model to which thecompound has been administered, the compound can be screened for as atleast one of the insulin resistance improving agent and the preventiveor therapeutic agent for diabetes. Notably, the HOMA-IR value can becalculated by the above-described HOMA-IR method.

<Other Steps>

The other steps are not particularly limited and may be appropriatelyselected depending on the intended purpose. In one exemplary step, usingas an index other diabetes-related factors and the like, furtherscreening is performed on the compounds having made the HOMA-IR value ofthe diabetes model mouse lower than the HOMA-IR value (X) of the controlin the above evaluation, to thereby select a compound effective as theinsulin resistance improving agent and the preventive or therapeuticagent for diabetes.

EXAMPLES

Hereinafter, the examples of the present invention will be specificallyexplained, but these examples shall not be construed as to limit thescope of the present invention.

Production Example 1 Synthesis of New Compound

The peptide expressed by the following SEQ ID No. 1 was used as the newcompound. The peptide expressed by the following SEQ ID No. 1 waschemically synthesized using a peptide synthesizer (product of ShimadzuCorporation).

(SEQ ID No. 1) Lys-Lys-Tyr-Ile-Leu-Ala-Leu

Test Example 1 Phosphorylation Inhibitory Activity

In the below-described manner, the peptide expressed by the above SEQ IDNo. 1 was evaluated for phosphorylation inhibitory activity againstphosphorylation of Ser727 of STAT3 by ERK2.

<Selection of Compound Having Phosphorylation Inhibitory Activity>—Mixture Preparation Step— —Preparation of Reaction Liquid—

The following ingredients were used at a composition indicated in Table1 to prepare a reaction liquid: 5× reaction buffer (250 mM tris-HCl (pH7.5), 50 mM magnesium chloride, 0.1% by mass bovine serum albumin (BSA)and 5 mM dithiothreitol (DTT)), Active ERK solution (0.1 mg/mL) (productof Biosource Inc.), STAT3 solution (0.2 mg/mL) (product of Abcam Inc.)and distilled water.

TABLE 1 Reagents Amount added (μL) 5 × Reaction buffer 21 Active ERKsolution 1 STAT3 solution 25 Distilled water 13 Total 60

—Preparation of Compound—

The compound used was the peptide chemically synthesized in ProductionExample 1 (SEQ ID No. 1). Also, 5-iodotubercidin (product of CalbiochemInc.) was used as a control compound.

The peptide (SEQ ID No. 1) indicated in Production Example 1 wassuspended in dimethylsulfoxide (DMSO) so that the final concentrationthereof was adjusted to 10 mM.

The 5-iodotubercidin was suspended in DMSO so that the finalconcentration thereof was adjusted to 5 mM.

—Preparation of Mixture—

A 5×ATP-MgCl₂ solution (5 mM ATP, 50 mM magnesium chloride),dimethylsulfoxide (DMSO) and, as the above compound, the peptide (SEQ IDNo. 1) or 5-iodotubercidin were added to the above reaction liquid inthis order. Furthermore, distilled water was added to the samples so asto appropriately adjust the volume thereof, whereby reaction mixtureswere obtained.

Notably, the reaction mixtures each containing neither the peptide (SEQID No. 1) nor the 5-iodotubercidin were used as a control.

Each sample was prepared according to the following Table 2.

TABLE 2 3 1 2 1 mM 4 Control Control 5-Iodotubercidin 1 mM PeptideSample (22% DMSO) (10% DMSO) (22% DMSO) (10% DMSO) Reaction liquid (μL)5 5 10 10 5 × ATP-MgCl₂ 1.75 1.75 3.5 3.5 solution (μL) DMSO (μL) 2 0.850 0 10 mM peptide (μL) — — — 1.75 5 mM 5-iodotubercidin — — 4 — (μL)Distilled water (μL) 0 1.15 0 2.25 Total (μL) 8.75 8.75 17.5 17.5

From Table 2, the final concentration of the STAT3 is about 0.048 μg/mL,the final concentration of Active ERK2 is about 0.001 μg/mL, the finalconcentration of DMSO is 10% by mass, the final concentration of thepeptide is 1 mM and the final concentration of 5-iodotubercidin is 1.14mM.

Each of the reaction mixtures was incubated at 30° C. for 130 min toallow Active ERK2 to phosphorylate the Ser727 of the STAT3.

After that, to terminate the phosphorylation, 0.5M ethylenediaminetetraacetate (EDTA) was added in an amount of 2.75 μL to the reactionmixtures serving as a control (Table 2: Sample 1 or 2), and was added inan amount of 5.5 μL to the reaction mixture containing the5-iodotubercidin (Table 2: Sample 3) or the reaction mixture containingthe peptide (Table 2: Sample 4).

—Antibody Reaction Step— —Western Blotting—

Using an aliquot (23 μL) of each of Samples 1 to 4 indicated in Table 2after completion of the reaction, 4×SDS loading buffer (125 mM tris-HCl(pH 6.8), 4.3% by mass SDS, 30% by mass glycerol, 10% by mass2-mercaptoethanol and 0.01% by mass bromophenol blue) was added in anamount of 4 μL to the aliquot of the reaction mixture serving as thecontrol (Sample 1 or 2) and was added to in an amount of 8 μL to thealiquot of the reaction mixture containing the 5-iodotubercidin (Sample3) or the aliquot of the reaction mixture containing the peptide (Sample4), followed by boiling at 95° C. for 5 min.

Each (15 μL) of the above samples was applied to MULTIGEL (product ofDaiichi Kagaku Yakuhin Co., Ltd.), where the sample waselectrophoresized for 30 min at a constant current of 30 mA in anelectrophoresis buffer (25 mM tris-HCl (pH 8.4), 192 mM L-glysine, 0.1%by mass SDS).

After completion of the electrophoresis, the MULTIGEL was washed withdistilled water for 10 min.

The MULTIGEL was sufficiently immersed in a transfer buffer (25 mMtris-HCl, 192 mM L-glysine, 20% by mass methanol). Then, throughblotting for 60 min at a constant current of 10 V, the electrophoresizedproteins were transferred from the MULTIGEL to a PVDF membrane(Hybond-P, produce of GE Healthcare).

—Primary Antibody Reaction—

The primary antibody reaction was performed as follows using SNAPid(product of Millipore Inc.).

Specifically, skimmed milk (product of Snow Brand Milk Products Co.,Ltd.) was suspended in TBS (20 mM tris-HCl (pH 7.5), 140 mM sodiumchloride) so as to have a concentration of 0.5% by mass. After thethus-prepared 0.5% by mass skimmed milk (10 mL) had been added to thewells of the SNAPid, the PVDF membrane having the transferred proteinswas subjected to blocking while being drawn with a vacuum pump.

Next, 9 μL of anti-p5727 STAT (Cat. No.: sc-21876, product of SANTA CRUZInc.) used as the primary antibody was suspended in 3 mL of TBST (20 mMtris-HCl (pH 7.5), 140 mM sodium chloride, 0.05% by mass tween-20).After removal of the blocking solution, the primary antibody was addedto the wells, followed by being left to stand still for 10 min, tothereby allow the primary antibody to bind to the STAT3 whose Ser727 hadbeen phosphorylated.

After removal of the solution containing the primary antibody, 10 mL ofTBST was added to the wells to wash the unbound primary antibody. Thewashing of the primary antibody was performed three times.

—Secondary Antibody Reaction—

The secondary antibody reaction was performed as follows using theSNAPid.

First, 16.2 μL of Goat anti-rabbit IgG AP conjugated (product ofCalbiochem Inc., Cat. No.: DC06L) used as the secondary antibody wassuspended in 3 mL of TBST. After removal of the TBST used for washingthe primary antibody, the secondary antibody was added to the wells,followed by being left to stand still for 10 min, to thereby allow thesecondary antibody to bind to the primary antibody.

Next, after removal of the solution containing the secondary antibody,10 mL of TBST was added to the wells to wash the unbound secondaryantibody. The washing of the secondary antibody was performed threetimes.

—Color-Developing Reaction—

The PVDF membrane having undergone the secondary antibody reaction waswashed for 5 min with a color-developing buffer (0.1M tris-HCl (pH 9.5),0.1M sodium chloride, 50 mM magnesium chloride).

Then, the PVDF membrane was immersed in a color-developing reactionliquid (10 mL) for color-developing reaction. After color-developingreaction for several minutes, the color development was visuallyconfirmed, followed by washing with TBST.

Notably, the color-developing reaction liquid was prepared as follows.Specifically, 45 μL of a NBT solution (which had been prepared bysuspending 173 mM nitroblue tetrazolium in 70% by massdimethylformamide) and 35 μL of a BCIP solution (which had been preparedby suspending 250 mg bromochloroindolylphosphate(5-bromo-4-chloro-3-indolyl phosphate) in 5 mL of dimethylformamide(DMF)) were mixed together to obtain the color-developing reactionliquid.

The whole image obtained by the western blotting is given in FIG. 1, andthe enlarged view of FIG. 1 is given in FIG. 2. In FIGS. 1 and 2, fromthe right to the left, “M” corresponds to a lane of a molecular-weightmarker, “1” corresponds to a lane of Sample 1 serving as a control ofthe 5-iodotubercidin, “2” corresponds to a lane of Sample 2 serving as acontrol of the peptide expressed by SEQ ID No. 1, “3” corresponds to alane of Sample 3 containing the 5-iodotubercidin, and “4” corresponds toa lane of Sample 4 containing the peptide expressed by SEQ ID No. 1.

—Screening Step—

From the result of the western blotting in Test Example 1, the band wasdetected in the 5-iodotubercidin (Sample 3) but was lower in intensitythan the band detected in the control (Sample 1). Thus, it was confirmedthat the 5-iodotubercidin had a phosphorylation inhibitory activity. Incontrast, no band was detected in the peptide expressed by SEQ ID No. 1(Sample 4). Thus, it was thought that this peptide had a phosphorylationinhibitory activity higher than that of the 5-iodotubercidin (Sample 3).

Therefore, it was recognized that the peptide expressed by SEQ ID No. 1was useful as a phosphorylation inhibitor for the phosphorylation of theSer727 of the STAT3.

Test Example 2 Acute Toxicity Test

The peptide expressed by SEQ ID No. 1 was examined for acute toxicity asfollows.

<Test Animal> —Acclimation—

Fifty-four female db/db mice (body weight: 40 g to 60 g, age: 56 daysold to 70 days old, product of Shanghai SLAC Laboratory Animal Inc.)were used as the test animal.

After arrival of the above mice, they were evaluated for general healthconditions and acclimated for 7 days before test.

—Breeding—

The mice were kept as a population during acclimation. Then, duringtheir lives, they were individually kept according to the guideline ofthe U.S. National Research Council “Guide for the Care and Use ofLaboratory Animals.”

The environment of the animal room was controlled to a temperature of18° C. to 26° C. and a relative humidity of 30% to 70%. In addition,each of the daytime and the nighttime was controlled to become 12 hours.The temperature and relative humidity were measured every day.

Regarding feeding, feed (product of Shanghai SLAC Laboratory Animalinc., Cat. No.: M-01F) was given to all the test animals ad libitum.

On the previous day before the measurement of the fasting blood glucoselevel and the blood insulin level, they were fed during a period of20:30 to 21:00. After the above period had passed, the remaining feedwas removed, if any. The fasting period was set to 12 hours to 14 hours,and the test animals were fed after blood sampling. After subjected tohigh-pressure sterilization, water was given to them ad libitum.

—Selection of Test Animal—

On the basis of the blood glucose levels measured during feeding andfasting, 52 test animals were selected out of the 54 test animals. Thetest animal exhibiting too high or low a blood glucose level wasexcluded.

—Clinical Remarks—

Once a day, each of the test animals was confirmed for survival andstool in the morning, and was observed for clinical remarks in theafternoon.

<Acute Toxicity Test> —Method—

Two individuals of the above mice were used to study the peptideexpressed by SEQ ID No. 1 for acute toxicity.

The above peptide was treated in the following manner. Specifically, 8.0mg of the peptide was dissolved in 0.4 mL of physiological saline toprepare a 20 mg/mL peptide solution (hereinafter may be referred to as“peptide solution 1”).

The peptide solution 1 was newly provided before administration, andstirred so that the peptide was dissolved completely and homogeneously.

The above-prepared peptide solution 1 was intraperitoneally injectedonce to one mouse at a dose of 30 mg/kg and to the other mouse at a doseof 100 mg/kg.

The mice having received the peptide solution 1 were monitored forclinical signs and change in body weight for 14 days. The fasting bloodglucose level and fasting blood insulin level were measured on Day 0 andDay 14 by the below-described method.

—Sampling of Blood Sample—

To measure the fasting blood glucose level and blood insulin level, 50μL of blood was sampled from the postorbital region on Day 0 and Day 14after administration. To obtain serum, the blood sample was left tostand in a tube on ice and, within 1 hour, the blood sample wascentrifuged (5,000 g, 20 min, 2° C. to 8° C.). The fraction obtainedthrough the centrifugation was transferred to a polyethylenemicrocentrifuge tube, which was stored in a frozen state at −80° C.before analysis.

—Blood Chemical Analysis—

The blood glucose level was determined using Johnson one-touch UltraGlucose Monitoring System (product of Johnson & Johnson K.K.).

The blood insulin level was determined using a mouse insulin ELISA kit(Cat. #EZRMI-13K, product of Linco Research Inc.).

—Results—

The results of the acute toxicity test are given in the following Table3.

TABLE 3 Day 0 Day 14 Fasting Fasting Fasting blood blood blood Bodyglucose insulin Body Fasting blood insulin Dose weight level levelweight glucose level level (mg/kg) (g) (mM) (ng/mL) (g) (mM) (ng/mL) 3042.53 22.7 18.78 44.6 15.3 17.68 100 43.36 18.3 13.95 44.5 25.3 12.85

From Table 3, the mouse having received the above peptide solution 1 ata dose of 30 mg/kg or 100 mg/kg exhibited no significant changes in thefasting blood glucose level, the fasting blood insulin level and thebody weight. In addition, there were no adverse events observed from theclinical remarks. The fasting blood glucose level and the fasting bloodinsulin level on Day 0 and Day 14 after administration are given inTable 3.

Test Example 3 Efficacy Test

The compound exhibiting a phosphorylation inhibitory activity in TestExample 1 (i.e., the peptide expressed by SEQ ID No. 1) was evaluatedfor efficacy as at least one of an insulin resistance improving agentand a preventive or therapeutic agent for diabetes in the followingmanner.

<Method> —Test Animal—

Fifty mice described in Test Example 2 were used as a test animal.

—Preparation of Peptide Solution and Metformin Solution—

To study the efficacy of the peptide expressed by SEQ ID No. 1, apeptide solution was prepared as follows.

Specifically, 3.8 mg of the above peptide was dissolved in 4.75 mL ofphysiological saline to prepare a 0.8 mg/mL peptide solution(hereinafter may be referred to as “peptide solution 2”). Then, 1.75 mLof the peptide solution 2 was dissolved in 3.5 mL of physiologicalsaline to prepare a 0.27 mg/mL peptide solution (hereinafter may bereferred to as “peptide solution 3”). Further, 1 mL of the peptidesolution 3 was dissolved in 2 mL of physiological saline to prepare a0.09 mg/mL peptide solution (hereinafter may be referred to as “peptidesolution 4”).

A metformin solution used for an activity control group was prepared asfollows. Specifically, 120 mg of metformin (product of Sigma Co.) wasdissolved in 4 mL of physiological saline to prepare a 30 mg/mLmetformin solution.

Each of the peptide solutions 2 to 4 and the metformin solution wasnewly provided before administration and stirred so that the peptide ormetformin was dissolved completely and homogeneously.

—Administration—

The efficacy test was conducted using the following five administrationgroups: a solvent (physiological saline) control administration group, a150 mg/kg metformin administration group (serving as an activity controlgroup), a 4.00 mg/kg peptide (expressed by SEQ ID No. 1) administrationgroup, a 1.33 mg/kg peptide administration group and a 0.44 mg/kgpeptide administration group. Each administration group had 10 mice.

The above-prepared metformin solution was used for the 150 mg/kgmetformin administration group, the peptide solution 2 was used for the4.00 mg/kg peptide administration group, the peptide solution 3 was usedfor the 1.33 mg/kg peptide administration group, and the peptidesolution 4 was used for the 0.44 mg/kg peptide administration group.

The appropriate dose of metformin administered to human is 750 mg/day.This dose was converted based on the body weight of mouse, and metforminwas administered to the mice at an excessive amount of 150 mg/kg.

The above-prepared solutions were intraperitoneally injected every dayto all of the mice of the test groups at the corresponding doses givenin the following Table 4 for 28 days. The body weight, the fasting bloodglucose level, the postprandial blood glucose level and the fastingblood insulin level were measured by the below-described measuringmethod according to the measurement schedule given in the followingTable 5.

Notably, as indicated in Table 6, the above 50 mice were divided intofive administration groups so that the distribution of each of thefasting blood glucose level, the fasting blood insulin level and theHOMA-IR value (index of insulin resistance) (measured on Day 0) becameequal between the administration groups. The calculating method for theHOMA-IR value is described below.

FIG. 3 gives the fasting blood glucose level on Day 0 in eachadministration group.

FIG. 5 gives the fasting blood insulin level on Day 0 in eachadministration group.

FIG. 6 gives the HOMA-IR value on Day 0 in each administration group.

—Sampling of Blood Sample—

To measure the fasting blood glucose level and blood insulin level, 50μL of blood was sampled from the postorbital region on Day 0, Day 14 andDay 28 after administration. The serum was obtained in the same manneras in Test Example 2.

The postprandial blood glucose level was measured using blood sampledfrom the tail vein on Day 1, Day 13, and Day 27 after administration.

—Blood Chemical Analysis—

The blood glucose level and blood insulin level were measured in thesame manner as in Test Example 2.

The insulin resistance was determined by the HOMA (homeostasis modelassessment) method using the following calculation formula.

HOMA-IR (index of insulin resistance)=fasting insulin level(μU/mL)×fasting blood glucose level (mmol/L)/22.5

Here, it can be recognized that the lower the HOMA-IR value is, the moreimproved the insulin resistance is (see Matthews D R et al.,Diabetologia 1985 July 28(7), 412-9, Kanauchi M et al., Diabetes Care,October 25(10), 2002, p. 1891-2).

—Numerical Analysis—

All of the values are indicated as “average value±average error.” Theimportant differences between the groups and within each group wereevaluated through one-way variance analysis and Dunnett's multiplecomparison. The p-values equal to or lower than 0.05 were regarded asstatistically significant.

TABLE 4 Volume Administration Administration Dose administeredconcentration Test group Number/Sex method (mg/kg) (mL/kg) (mg/mL)Solvent 10/Female Intraperitoneal NA 5 NA control injection Metformin10/Female Intraperitoneal 150 5 1.00 (150 mg/kg) injection Peptide10/Female Intraperitoneal 4.00 5 0.80 (4.00 mg/kg) injection Peptide10/Female Intraperitoneal 1.33 5 0.27 (1.33 mg/kg) injection Peptide10/Female Intraperitoneal 0.44 5 0.09 (0.44 mg/kg) injection

TABLE 5 Measurement method Measurement days Body weight 0, 7, 14, 21, 28Fasting blood glucose level 0, 14, 28 Postprandial blood glucose level1, 13, 27 Fasting blood insulin level 0, 28

TABLE 6 Post- Fasting prandial Fasting blood blood blood glucose glucoseinsulin level level level HOMA-IR Test group (mM) (mM) (ng/mL) valueSolvent 14.4 ± 1.3 18.2 ± 1.9 9.23 ± 1.26 149.0 ± 20.0 control Metformin16.3 ± 1.3 22.5 ± 2.0 8.81 ± 1.33 139.4 ± 22.7 (150 mg/kg) Peptide 13.5± 1.5 23.0 ± 1.7 8.87 ± 0.69 149.1 ± 11.5 (4.00 mg/kg) Peptide 13.6 ±1.2 22.4 ± 1.6 9.05 ± 1.17 157.4 ± 19.2 (1.33 mg/kg) Peptide 14.1 ± 1.422.9 ± 1.5 8.45 ± 0.6 155.6 ± 16.6 (0.44 mg/kg)

<Results>

The following Table 7 gives the postprandial blood glucose levelsmeasured on Day 13 after administration and the fasting blood glucoselevels measured on Day 14.

FIG. 4 gives the postprandial blood glucose levels measured on Day 13after administration in each administration group.

FIG. 3 gives the fasting blood glucose levels measured on Day 14 afteradministration in each administration group.

TABLE 7 Fasting blood Postprandial glucose blood level glucose Testgroup (mM) level (mM) Solvent control 17.6 ± 1.1 26.1 ± 2.0 Metformin19.3 ± 1.5 25.2 ± 2.0 (150 mg/kg) Peptide 15.2 ± 1.2 30.3 ± 0.9 (4.00mg/kg) Peptide 17.4 ± 1.3 31.0 ± 0.7 (1.33 mg/kg) Peptide 16.5 ± 1.127.9 ± 0.8 (0.44 mg/kg)

The following Table 8 gives the HOMA-IR values calculated from thepostprandial blood glucose levels measured on Day 27 afteradministration and the fasting blood glucose levels and the fastingblood insulin levels measured on Day 28 after administration.

FIG. 4 gives the postprandial blood glucose levels measured on Day 27after administration in each administration group.

FIG. 3 gives the fasting blood glucose levels measured on Day 28 afteradministration in each administration group.

FIG. 5 gives the fasting blood insulin levels measured on Day 28 afteradministration in each administration group.

FIG. 6 gives the fasting HOMA-IR values measured on Day 28 afteradministration in each administration group.

Notably, “#” in FIGS. 3, 5 and 6 means that the values indicated therebyare statistically significant with respect to the solvent control groupas a result of the numerical analysis.

TABLE 8 Post- Fasting prandial Fasting blood blood blood glucose glucoseinsulin level level level HOMA-IR Test group (mM) (mM) (ng/mL) valueSolvent control 22.5 ± 1.2 29.7 ± 0.8 6.00 ± 0.98 147.3 ± 23.6 Metformin 22.0 ± 3.1 28.1 ± 1.6 4.06 ± 0.84  79.6 ± 17.7^(#) (150 mg/kg)Peptide 22.3 ± 1.0 28.3 ± 1.0  3.36 ± 0.41^(#)  71.4 ± 8.6^(#)  (4.00mg/kg) Peptide 21.5 ± 1.7 30.1 ± 1.3  3.59 ± 0.79^(#)  76.1 ± 17.3^(#)(1.33 mg/kg) Peptide  19.3 ± 1.1^(#) 29.4 ± 0.8 4.23 ± 1.12 79.8 ± 21.4(0.44 mg/kg) ^(#)p < 0.05 vs. solvent control group

The difference between the fasting blood glucose levels, thepostprandial blood glucose levels or the fasting blood insulin levelsmeasured on Day 28 and Day 0 after administration was calculated fromthe following calculation formulas as an increment of each level. Theresults are given in the following Table 9.

Increment in fasting blood glucose level=fasting blood glucose level onDay 28—fasting blood glucose level on Day 0

Increment in postprandial blood glucose level=postprandial blood glucoselevel on Day 28—postprandial blood glucose level on Day 0

Increment in fasting blood insulin level=fasting insulin level on Day28—fasting insulin level on Day 0

FIG. 7 gives the increments in fasting blood glucose level andpostprandial blood glucose level in each administration group.

FIG. 8 gives the increment in fasting blood insulin level in eachadministration group.

Notably, “#” in FIG. 7 means that the value indicated thereby isstatistically significant with respect to the solvent control group as aresult of the numerical analysis.

TABLE 9 Increment in Increment in Increment in fasting bloodpostprandial fasting blood glucose level blood glucose insulin levelTest group (mM) level (mM) (ng/mL) Solvent control 11.1 ± 1.7 11.5 ±1.9  −3.94 ± 1.64 Metformin  5.8 ± 2.4  4.3 ± 2.2^(#) −4.94 ± 1.77 (150mg/kg) Peptide  8.8 ± 1.4 5.3 ± 1.4 −3.51 ± 0.96 (4.00 mg/kg) Peptide 7.9 ± 1.5 7.7 ± 1.4 −5.52 ± 1.33 (1.33 mg/kg) Peptide  5.7 ± 1.8 6.7 ±2.2 −4.12 ± 1.3  (0.44 mg/kg) ^(#)p < 0.05 vs. solvent control group

As indicated in Tables 6 to 9, the increment of the fasting bloodglucose level in the solvent control group was 11.1±1.7 mM, while theincrement of the fasting blood glucose level in the 150 mg/kg metforminadministration group was 5.8±2.4 mM. Thus, metformin suppressed increasein fasting blood glucose level from Day 0 to Day 28 (Table 9, FIG. 7).However, the suppression of increase in fasting blood glucose level inthe 150 mg/kg metformin administration group was not statisticallysignificant.

Meanwhile, the fasting blood insulin level measured on Day 28 afteradministration decreased in the 150 mg/kg metformin administration group(Table 8, FIG. 5), resulting in that the HOMA-IR value (index of insulinresistance) was significantly decreased (Table 8, FIG. 6).

Also in the administration groups of the peptide expressed by SEQ ID No.1, similar to metformin, the suppressions of increase in fasting bloodglucose level were not statistically significant for a test period of 28days (Table 9, FIG. 7). However, in each of the 1.33 mg/kg peptideadministration group and the 4.00 mg/kg peptide administration group,the fasting blood insulin level (Table 8, FIG. 5) and the HOMA-IR value(Table 8, FIG. 6) measured on Day 28 after administration significantlydecreased as compared with the solvent control group.

Also, in the administration groups of the peptide expressed by SEQ IDNo. 1, the fasting blood insulin level and the HOMA-IR level decreasedin a dose-dependent manner (Table 8).

Furthermore, although the dose of the 1.33 mg/kg peptide administrationgroup or the 4.00 mg/kg peptide administration group was lower than thatof the 150 mg/kg metformin administration group, the fasting bloodinsulin level and the HOMA-IR level decreased as compared with the 150mg/kg metformin administration group (Table 8, FIGS. 5 and 6).

These results indicate that the peptide expressed by SEQ ID No. 1 iseffective as at least one of an insulin resistance improving agent and apreventive or therapeutic agent for diabetes.

Also, since the effective concentration of the peptide expressed by SEQID No. 1 is lower than that of the existing drug metformin, the peptideis advantageously used as at least one of an insulin resistanceimproving agent and a preventive or therapeutic agent for diabetes.

All examples and conditional language recited herein are intended forpedagogical purposes to aid the reader in understanding the inventionand the concepts contributed by the inventor to furthering the art, andare to be construed as being without limitation to such specificallyrecited examples and conditions, nor does the organization of suchexamples in the specification related to a showing of the superiorityand inferiority of the invention. Although the embodiments of thepresent invention have been described in detail, it should be understoodthat the various changes, substitutions, and alterations could be madehereto without departing from the spirit and scope of the invention.

INDUSTRIAL APPLICABILITY

First, the disclosed new compound inhibits phosphorylation of Ser727 ofSTAT3 to exhibit an excellent phosphorylation inhibitory activityagainst phosphorylation of Ser727 of STAT3, and thus can suitably beused as a phosphorylation inhibitor for Ser727 of STAT3. In addition,the phosphorylation inhibitor has a target molecule and mechanism whichare for reducing the blood glucose level of patients with diabetes andwhich are different from those of existing drugs, and thus is useful asa preventive or therapeutic agent for insulin resistance, diabetes,obesity, abnormal lipid metabolism, high blood pressure and otherpathological conditions. The phosphorylation inhibitor is useful notonly as a drug but also as a reagent for assay using phosphorylation asan index.

Second, the disclosed screening method can suitably used for screeningfor at least one of the insulin resistance improving agent and thepreventive or therapeutic agent for diabetes.

1. A screening method for at least one of an insulin resistanceimproving agent and a preventive or therapeutic agent for diabetes, thescreening method comprising: determining the presence or absence ofinteraction between STAT3 and ERK2 to select a compound having aphosphorylation inhibitory activity, and evaluating the compound forwhether X>Y where X denotes a HOMA-IR value of a diabetes model animalto which the compound has not been administered and Y denotes a HOMA-IRvalue of a diabetes model animal to which the compound has beenadministered.
 2. The screening method according to claim 1, wherein thedetermining is performed by preparing a mixture by mixing together atleast the compound, the STAT3 whose Ser727 is not phosphorylated, and anenzyme that phosphorylates the STAT3; reacting the STAT3 whose Ser727has been phosphorylated in the mixture with an antibody that binds to aphosphorylated site of the STAT3 whose Ser727 has been phosphorylated;and screening for the compound present in the mixture containing theSTAT3 to which the antibody has not bound, as a compound having thephosphorylation inhibitory activity.
 3. The screening method accordingto claim 2, wherein the preparing comprises phosphorylating the Ser727of the STAT3 contained in the mixture.
 4. The screening method accordingto claim 2, wherein the reacting comprises detecting the antibodybinding to the phosphorylated site of the STAT3 whose Ser727 has beenphosphorylated.
 5. The screening method according to claim 4, whereinthe detecting is performed with IgG derived from a host of the antibodybinding to the phosphorylated site of the STAT3 whose Ser727 has beenphosphorylated.
 6. The screening method according to claim 5, whereinthe IgG is detected with a label bound thereto.
 7. The screening methodaccording to claim 2, wherein the screening is performed by determiningthe presence or absence of the phosphorylation inhibitory activity ofthe compound.